Malignant melanoma is a form of skin cancer that can develop from melanocytes, cells that are capable of forming melanin, a dark-brown to black pigment. Most melanomas develop from a previous mole over a period of several months or years and occur most commonly in fair-skinned people that are intolerant to sunlight. There are several distinct types of melanomas. Prognosis depends on the kind of melanoma, its depth of invasion, its location, and the age and health of the patient.
According to new statistics, malignant melanoma could become a lethal epidemic in the next decade. In 1993, 32,000 people in the United States developed melanoma, and 6,420 died from it. Exactly why the rate of skin cancer is growing so rapidly is not known although accumulating evidence suggests that sun exposure and the thinning ozone layer play a major role.
Patients who receive early diagnosis and treatment have a high chance of survival. Most patients in the early stages of melanoma undergo surgery to remove the primary tumor. Patients with locally advanced or metastatic disease usually also undergo some type of chemotherapy and may also receive radiation therapy although radiation is not usually very effective against melanoma. Other treatments include cytotoxic drugs, treatment with immunological substances and autologous (self) bone marrow transplant to enhance the patient's immune system.
Melanoma surgical care is undergoing change with the advent of pre-operative and intra-operative mapping techniques that identify the sentinel node of the lymphatic basin. Lymphoscintigraphy performed on a group of patients in a recent study [Redefinition of Cutaneous Lymphatic Drainage with the Use of Lymphoscintigraphy for Malignant Melanoma; Norman, J, Reintgen, D S, et al.; Amer. J. Surg.; 162; November 1991; 432 (6)] has demonstrated that the lymphatic drainage pathways identified in classical anatomical studies differ significantly from the results obtained from in vivo models. The new study demonstrates that there are much larger areas of ambiguous drainage than previously reported. Further, the study demonstrates a high level of individuality of cutaneous lymphatic flow. It is estimated that over 50% of all lymph node dissections for intermediate thickness melanomas may be misdirected if pre-operative mapping is not performed.
Histopathologists usually examine and determine the extent of melanomas of the skin by measuring the thickness of the tumor and the level of penetration into the skin measured in millimeters from the top of the granular cell layer to the deepest point of tumor extension. Breslow [Breslow, A., Tumour Thickness, Level of Invasion and Node Dissection in Stage 1 Cutaneous Melanoma, Ann. Surg 1975; 182: 572-75] has developed a commonly used grading system for melanoma thickness. This is regarded as the most important morphological variable for determining prognosis in patients with localized disease. Breslow grouped tumor thickness into three categories; thin [&lt;0.76 mm], intermediate [0.76 mm-4.00 mm] and thick [&gt;4.00 mm]. Recent reports however, indicate certain problems associated with assessing Breslow thickness [Measuring melanomas (editorial) The Lancet; 338; Aug. 10, 1991; 351(2)]. Level of invasion, classified by Clark [Clark, W. H. Jr., From, L.; Bernardino, A.; Mihm, M. C.; The Histogenesis and Biological Behaviour of Primary Human Malignant Melanomas of the skin; Cancer Res. 1969; 29; 705-26] into five categories on the basis of the normal anatomy of the dermis carries less prognostic weight but is usually also included in histopathological reports.
Melanoma may be localized (Stage 1 and 2 disease) or the cancer may have spread (metastasized) to other parts of the body (Stage--3 or 4 disease). Researchers have long sought a quick, simple to use, inexpensive test to determine the presence of metastatic melanoma.
There are a number of methods that are used in the clinical laboratories to determine whether the melanoma is metastatic. Currently available tests are based on the light microscopic examination of samples of bone marrow, lymph tissues or other tissue samples.
Morphological distinctions exist between tumor cells and lymphocytes (white blood cells found in large numbers in the blood and the lymph). However, it can be difficult to distinguish melanoma cells from normal lymphocytes or other normal cells, especially when only a small proportion of the cells are abnormal or the aberrant cell is not apparent. The problem becomes even more severe when the melanoma is in the early stages of metastasis and only a very small number of tumor cells are present in the blood, lymph node or bone marrow.
Another problem with the standard procedure for examining lymph nodes for tumor involvement is the quantity of tissue removed for sampling. The examination of 1 or 2 sections from the center of the lymph node samples only about 1/1000 of the tissue submitted for pathological examination.
Routine histological examination and histologic staining for the evaluation of metastatic melanoma are currently available in many hospitals. However, histopathological examination is not sufficiently sensitive to adequately diagnose early metastasis of melanoma. False negative results may be obtained because of very few tumor cells in the pathologic samples. This is borne out by the fact that approximately 25 to 33 percent of the patients who are histologically node negative will have recurrent tumors and die of their disease. Despite good prognostic factors, metastases are missed and patients die of their disease.
The histopathological approach routinely underestimates the number of patients with metastases. The standard histopathology interpretation with hematoxylin-eosin staining (H&E staining and light microscopy evaluation) routinely used for the detection of metastatic tumor cells in tissue has a sensitivity of 1 in 10.sup.4 normal cells. The rate limiting factor of routine histopathology examination is the number of sections of the lymph node made, stained and examined; the examination becomes limited when only 1/1000 of the tissue submitted for processing is ordinarily examined.
Methods such as serial sectioning and immunohistochemistry are used to increase the sensitivity of the tests. Recently, marker tests relying on antibodies specific to components on the cell surface of tumor cells have been developed. In general, these antibodies react with glycoproteins or gangliosides on the surface of the target cell. When such glycoproteins or gangliosides are present in abnormally large amounts or are present in conditions where they are not usually found, they are a signal that abnormal cells are present or are circulating.
If the tissue being evaluated for the presence of metastatic melanoma appears to be pathologically suspicious upon inspection, immunohistochemical staining with antibodies against S-100 protein or HMB-45 melanoma antigen can help to confirm the diagnosis. Unfortunately, these markers are not unique to cancer cells and their usefulness depends upon what is tested and the number of sections examined.
Even though the techniques have been available for a number of years and have been reported to increase the yield of occult metastases by a factor of 2 these assays have not been incorporated into the routine screening of lymph node sections in the community. The time and expense involved prevents them from being used routinely and they will never be widely adopted.
An additional problem with these methods is that they are based on finding changes in morphologic features and/or expression of specific proteins, methods that are not always accurate and can lead to ambiguous results. Furthermore, even such immunohistological methods are not sufficiently sensitive to properly diagnose early metastasis of melanoma.
As stated above, most tests are based on the examination of samples of blood, lymph tissues or other tissues of the body to determine the presence of melanoma cells. The presence or absence of lymph node metastases in patients with malignant melanoma is the single most powerful prognostic factor for predicting survival.
Clinically, the management and the five year survival rate for Stage III melanoma patients with regional lymph node involvement are different from that of Stage I or II patients. Either immunotherapy or chemotherapy is needed besides surgical dissection. So the early detection of metastasis in lymph nodes is very important for the patient classification, treatment and prognosis.
As an example of the importance of proper staging, the 5 year survival for Stage 3 melanoma patients with regional nodal involvement is 50% decreased compared to the survival of node negative patients. Adjuvant therapies have been attempted in order to increase the survival of the Stage 3 melanoma patient who has no evidence of disease but where the disease is likely to recur. Theoretically, the accurate and early detection of metastatic disease would allow those Stage 3 patients to be enrolled in the adjuvant trials earlier when systemic tumor burden is small and the likelihood of success is better.
The minute quantities of target cells present in lymph tissue makes it imperative that more sensitive techniques of selectively increasing the population of target cells be used. One technique uses cell culture methods to increase the population of target cells. [Heller, Reintgen, et al., Arch. Surg. 1991:126:1455-1460]
In one novel cell culture method for detecting micrometastases, the entire node is sampled by placement in tissue culture, much different than routine histologic examination that samples at most 0.1% of the submitted tissue. With this sensitive cell culture technique, over twenty percent of Stage 1 and 2 melanoma patients can be upstaged to Stage 3 disease (nodal metastases) when compared to routine histologic examination. Patients who were histologically node negative but culture node positive had a poorer disease free survival when compared to those patients who were node negative by both methods. Although this test offers a more sensitive method than histologic examination, on average it takes 4-6 weeks to obtain definitive results and has limited applicability to the community hospital setting. Furthermore, the tissue culture technique is fairly laborious.
Since melanocytes are not normally present in peripheral blood, bone marrow or lymph nodes, the presence of such cells in these immune system components is an indication that there are metastatic melanoma cells present. In addition to the presence of the malignant cells themselves, parts or artifacts of the cells presence may also be utilized to determine the presence of the target cells. Thus the presence of mRNA demonstrating the active expression of genes not expressed by cells normally occurring in peripheral blood, bone marrow or lymph tissue or the presence of the protein itself could be utilized as the indicator of the presence of the disease.
These diagnostic techniques are dependent upon 1) the presence of markers such as cells, cell parts or cell proteins that are not present in the sample in the absence of disease and 2) a method, sufficiently sensitive and specific, of indicating the presence of the chosen marker.
Through the work of a series of experimenters it has now been determined that the presence of mRNA transcribed from the tyrosinase gene is a useful indicator that there are metastatic melanoma cells present.
Tyrosinase is a copper-based oxidoreductase that catalyzes the oxidation of tyrosinase to dopa and the oxidation of dopa to dopaquinone. These are the first two steps in the biosynthesis of melanin, the pigment in normal skin. Tyrosinase is a key and rate limiting enzyme during melanin synthesis in melanocytes and melanoma cells. Since tyrosinase is not normally found in lymph nodes, the presence of tyrosinase and, by extension, melanin containing cells in lymph node sections is good evidence that metastatic melanoma cells are present.
Another diagnostic technique would utilize the occurrence of active transcription of the tyrosinase gene as an indicator of the presence of metastatic melanoma cells. If messenger RNA for tyrosinase is found in the lymph node sample, then that finding would indicate the active transcription of the tyrosinase gene and the presence of tyrosinase which in turn would indicate the presence of a melanocyte.
The most significant problem associated with these techniques is that in each case the target is present in such minute quantities that it is difficult if not impossible to separate any signal resulting from the presence of the target from the background present in the test system.
The invention of the polymerase chain reaction has provided a technique for rapidly and easily increasing the quantity of a specific DNA segment from a mixture containing a multitude of different DNA segments. Since PCR works to multiply only DNA segments, the target to be amplified must be in the form of DNA.
In actively reproducing cells multiple copies of messenger RNA (mRNA) for proteins expressed by that cell are likely to be present. Thus, the tyrosinase mRNA is a candidate for a diagnostic test for the presence of melanocytes.
In order to utilize the mRNA of tyrosinase in a PCR reaction, the mRNA must be converted to DNA. This can be accomplished by using reverse transcriptase. Reverse transcriptase is an enzyme that is capable of reading a strand of RNA and producing a strand of complementary DNA (cDNA).
In 1991, Smith and colleagues reported a procedure using reverse transcriptase and polymerase chain reaction for determining the presence of melanoma cells in peripheral blood. [Smith, B. et al., Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction; Lancet 1991; 338; 1227-29]. The procedure is based on the principle that the haematogenous spread of cancer influences the outcome of the disease for most patients. Since the number of circulating tumor cells in the blood is very small techniques for their detection are required to be both highly sensitive and highly specific.
Smith et al.'s procedure targets a gene-specific product of genes which are active in melanocytes but not in the cells normally found in peripheral blood. The procedure uses reverse transcriptase to prepare complementary DNA from messenger RNA contained in peripheral blood samples and polymerase chain reaction to amplify the target, in this case the cDNA made from the mRNA for tyrosinase.
This procedure is a significant advance over prior methods but it has several problems associated with it. The procedure does not allow the clinician to localize the site of the metastases; the volume of circulating blood is so large that the concentration of tumor cells in peripheral blood is inherently minimized; and the test is not useful in patients with apparently localized disease in whom peripheral blood cells may be few or absent. In addition, the most common site of melanoma metastases is regional nodes and the clinical relevance of finding circulating tumor cells is blood has not been proven. For instance, studies have shown that tumor cells can be routinely found in the peripheral circulation during operative procedures, but since these patients have a similiar disease progression to those in which no tumor cells can be demonstrated, it is more an academic rather than a clinically relevant finding.
Most importantly, the Smith technique does not work on lymph tissue. When attempts were made to utilize the Smith process on lymph tissue the result was always positive even in the absence of melanoma (false positives).
Thus there continues to exist the need for a fast, effective test for determining the presence of metastasized melanoma. In addition there exists a need for determining the localization of the metastatic tumor, the involvement of the patients lymph system since the lymph system is the first site of metastatistic disease and the determination of the involvement of other regions of the body. There is also the need for a test to determine whether apparently localized disease has metastasized to the lymph system, to develop a highly sensitive method to detect micrometastases by examining lymph nodes for the presence of tyrosinase messenger RNA and to determine whether a metastatic tumor is present even though it has not yet lodged in other locations in the patients body. Yet another problem relates to determining the quantity of metastasized cells present in a sample as an indicator of the advancement of the disease.